m7G-quant-seq: Quantitative Detection of RNA Internal N7-Methylguanosine

Methods for the precise detection and quantification of RNA modifications are critical to uncover functional roles of diverse RNA modifications. The internal m7G modification in mammalian cytoplasmic tRNAs is known to affect tRNA function and impact embryonic stem cell self-renewal, tumorigenesis, cancer progression, and other cellular processes. Here, we introduce m7G-quant-seq, a quantitative method that accurately detects internal m7G sites in human cytoplasmic tRNAs at single-base resolution. The efficient chemical reduction and mild depurination can almost completely convert internal m7G sites into RNA abasic sites (AP sites). We demonstrate that RNA abasic sites induce a mixed variation pattern during reverse transcription, including G → A or C or T mutations as well as deletions. We calculated the total variation ratio to quantify the m7G modification fraction at each methylated site. The calibration curves of all relevant motif contexts allow us to more quantitatively determine the m7G methylation level. We detected internal m7G sites in 22 human cytoplasmic tRNAs from HeLa and HEK293T cells and successfully estimated the corresponding m7G methylation stoichiometry. m7G-quant-seq could be applied to monitor the tRNA m7G methylation level change in diverse biological processes.

. (A) m 7 G/A levels in synthetic RNA oligo after m 7 G-seq NaBH4 treatment and m 7 G-quant-seq KBH4 treatment, versus the untreated input, revealed by LC-MS/MS. n = 3, technically independent replicates. (B) m 7 G/A levels in fragmented HeLa total RNA after m 7 G-seq NaBH4 treatment and m 7 G-quant-seq KBH4 treatment, versus the untreated input, revealed by LC-MS/MS. n = 3, biologically independent replicates. (C) The misincorporation or variation ratios at 18S rRNA m 7 G1639, which were uncovered by m 7 G-seq and m 7 G-quant-seq with total RNA isolated from wild-type or shControl HeLa cells. (D) The variation signatures at the AP site generated from HeLa 18S rRNA m 7 G1639 under m 7 G-quant-seq treatment with engineered RT1306 and adjusted dNTP/dATP ratios. (E) The variation signatures at the AP site generated from HeLa 18S rRNA m 7 G1639 under m 7 G-quant-seq treatment with Proto-Script II RT and adjusted dNTP/dATP ratios. (F) The variation signatures at the AP site generated from HeLa 18S rRNA m 7 G1639 under m 7 G-quant-seq treatment with SuperScript II RT and adjusted dNTP/dATP ratios. (G) The variation signatures at the AP site generated from HeLa 18S rRNA m 7 G1639 under m 7 G-quant-seq treatment with SuperScript IV RT and adjusted dNTP/dATP ratios. Figure S2. IGV plot of internal m 7 G46 site in representative tRNAs from HeLa cells. The upper two rows in each panel are from 'input' samples, n = 2, biologically independent replicates; the lower two rows in each panel are from 'm 7 Gquant-seq' samples, n = 2, biologically independent replicates. For tRNA labeled with (+), these tRNAs locate on sense strand in human genome, in which A, C, G, and U are marked by green, blue, brown, and red colors, respectively. For tRNA labeled with (-), these tRNAs locate on antisense strand in human genome, in which A, C, G, and U are marked by red, brown, blue, and green colors, respectively. Figure S3. (A) The variation ratios in m 7 G-quant-seq, variation ratios in 'Input' (with any chemical treatment), and estimated m 7 G methylation fraction at all guanosine sites in HeLa 18S rRNA. (B) The identified m 7 G sites in tRNA from different human cell lines, for a comparison of m 7 G detection in m 7 G-quant-seq and TRAC-seq.

Cell culture
HeLa and HEK 293T cell lines were purchased from the American Type Culture Collection (ATCC). The HeLa cell line was grown in DMEM medium (Gibco, 11965) supplemented with 10% v/v FBS and 1% penicillin/streptomycin (Gibco). The HEK 293T cell line was maintained in DMEM (Gibco, 11995) with 10% FBS and 1% penicillin/streptomycin. Cells were cultured at 37 °C with 5.0% CO2 in a Heracell VIOS 160i incubator (Thermo Scientific).

Small RNA isolation
Cellular total RNA was isolated with TRIzol reagent (Invitrogen) following the manufacturer's protocol by isopropanol precipitation. The small RNA fraction (size < 200 nt) was further extracted from the purified total RNA using the mirVana miRNA Isolation Kit (AM1560, Invitrogen).

RNA Fragmentation:
The starting amount of RNA could be 200 ng before fragmentation. Then RNA Fragmentation Reagent (Invitrogen, AM8740) was used as 15X (add 1.0 µL buffer into 14 µL of RNA), which is originally 10X. The heating condition is 70 °C for 14 min, followed by Oligo Clean and Concentrator kit (OCC). Elute RNA to 22 µL (twice, 11 µL each time). Because we expect to capture the real methylation fraction of the internal m 7 G site in tRNA, we did not perform any AlkB demethylation treatment to erase m 1 A, m 3 C, or m 1 G methylations on tRNA; in this way, it is hard for HIV RT to read through the full-size tRNA. Fragmenting tRNAs into 30-50 nt will facilitate HIV RT to read through the shorter fragments generated from tRNAs, reflecting the actual m 7 G methylation fraction via variation signatures.

End repair:
Prepare the end repair reaction as follow: Save 2 µL as 'input'; use the rest for the following steps. Safe stop point: -80C for 1 week.

Reduction:
Put the eluted RNA in 10 µL RNase-free water. Prepare a fresh 1.0M KBH4 buffer in RNase-free water. Add 40 µL KBH4 buffer into RNA and mix well. Incubate at room temperature for 4 hours (avoid the light). Recover RNA with RNA Clean and Concentrator kit (RCC) and elute to 45 µL with RNase-free water.
Prepare the ligation reaction as follow: Incubate the reaction at 25 °C for 12 hours.

PCR Amplification:
After the 12-hour cDNA ligation, heat at 65 °C for 5 min to denature. Purify the cDNA by DNA Clean & Concentrator-5. Elute cDNA with 20 µL DNase-free water. Use 4 µL per sample for each PCR amplification.

Identification of variation signatures in m 7 G-quant-seq
The sequencing data were all trimmed with the cutadapt tool to remove adapters and low-quality reads (lengths shorter than 20 bp). PCR duplicates were removed with the BBMap tool (https://sourceforge.net/projects/bbmap/), random barcodes at reads end were trimmed, and low-quality reads were removed using the cutadapt tool. The remaining reads were aligned to the human genome (hg38) using Tophat2 (version 2.1.1) and bowtie2 (version 2.3.5.1) allowing a maximum of three mismatches. The generated .bam files were split into positive and negative strands and sorted using Samtools. Sequence variants were identified by measuring the base composition at each position using fine-tuned bam-readcount (https://github.com/genome/bam-readcount). The generated bam-readcount output results were parsed and analyzed to calculate the misincorporation/deletion ratio at each abasic site generated from internal m 7 G site, followed by confirmation using direct visualization through IGV software (https://software.broadinstitute.org/software/igv/). The m 7 G candidate sites must satisfy the criteria shown below: (1) variation (misincorporation and deletion) ratio above 5% in m 7 G-quant-seq libraries; (2) variation ratio below 5% in 'Input' libraries; (3) total reads coverage depth above 20 in both m 7 G-quant-seq and 'Input' libraries; (4) variation ratio in m 7 G-quant-seq libraries is > 5-fold over that in 'input' libraries; (5) variation ratio in m 7 G-quant-seq libraries is > 5-fold over the background in any given sequence motif (defined as the variation rates detected from RNA probes containing unmodified NNGNN after m 7 G-quant-seq treatment). Additionally, all misincorporation and deletion signatures must occur at the internal positions of the reads, instead of reads end.
Note that the estimated m 7 G methylation fraction (in m 7 G -quant-seq) serves as the minimum value of methylation stoichiometry at the methylated site, because the calibration curves were built with NN(APsite)NN oligos instead of NN(m 7 G)NN oligos. The actual m 7 G methylation fraction could be a little higher than the estimated methylation stoichiometry in m 7 G -quant-seq, due to the fact that it is hard to achieve 100% chemical conversion at m 7 G site in all motif contexts.
Mix everything well before adding T4 RNA Ligase 1. Then add 1.0 µL T4 RNA Ligase 1 and mix well again. Add ligase enzyme to one sample followed by mixing this sample immediately. Mix each sample one by one.

S11
Incubate the reaction at 25 °C for 12 hours. After the 12-hour cDNA ligation, heat at 65 °C for 5 min to denature. Purify the cDNA by DNA Clean & Concentrator-5. Elute cDNA with 20 µL DNase-free water. Use 4 µL per sample for each PCR amplification.

Data availability
The sequencing data listed in Table S1 are available in the Gene Expression Omnibus database under the accession number GSE209646. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.  Duplicates for m 7 G-quant-seq "Treated" and "Input" samples to reveal tRNA m 7 G46 methylation fractions in HeLa and HEK293T cells, under HIV RT and 1mM dNTP.